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Second Trimester Inhibin A and New Approaches in Prenatal Screening

Second Trimester Inhibin A and New Approaches in Prenatal Screening. Jacob A. Canick, Ph.D. Women and Infants Hospital and Brown University Providence, Rhode Island, USA. Second Trimester Inhibin A and New Approaches in Prenatal Screening. Goals in prenatal screening Where are we now?

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Second Trimester Inhibin A and New Approaches in Prenatal Screening

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  1. Second Trimester Inhibin A andNew Approaches in Prenatal Screening Jacob A. Canick, Ph.D. Women and Infants Hospital and Brown University Providence, Rhode Island, USA J. Canick, 2003

  2. Second Trimester Inhibin A andNew Approaches in Prenatal Screening • Goals in prenatal screening • Where are we now? • New approaches: Addition of inhibin A • New approaches: The Integrated Test • New approaches: Fetal cells and DNA J. Canick, 2003

  3. Goals in Prenatal Screening • Improved test performance (make screening safer) • Optimal timing of test • Offer the best, safest diagnostic test • Make screening available to the largest number of women J. Canick, 2003

  4. Improving Screening Performance The challenge in screening is to have a test that has a high detection rate and low false positive rate. detection rate percentage of affecteds called screen positive by the test The higher the better! false positive rate percentage of unaffecteds called screen positive by the test The lower the better! J. Canick, 2003

  5. Optimal Timing of Screening Test • The earlier the better – only if it is also a better test! • How early do most women present for prenatal care? • What diagnostic test will be offered if a woman is screen positive? • Safer options if a serious fetal abnormality is identified J. Canick, 2003

  6. Offer the best, safest diagnostic test • For prenatal chromosome analysis, the choices are CVS or amniocentesis. • CVS is done no earlier than 10.5 weeks and usually not later than 13-14 weeks. • Amniocentesis is usually done no earlier than 14-15 weeks. • Is CVS easily available in your geographic area? • Which procedure is safer? • Which procedure has more errors? J. Canick, 2003

  7. Make prenatal screening available to the largest number of women • Ability to pay for test (government, insurance, self-pay?) • Geographic dispersion (in city, suburban area, rural area)? • Availability of quality laboratory services? • Availability of quality obstetrical ultrasound? • Availability of genetic counseling? • Availability of appropriate follow-up diagnostic procedures and tests? J. Canick, 2003

  8. Where are we now? J. Canick, 2003

  9. Maternal Serum Markers in the 2nd Trimester:Major improvement in screening for Down syndrome Background 1980s Maternal serum AFP already in use in screening for open NTDs. 1984 Maternal serum AFP is low in Down syndrome pregnancy. 1987 Maternal serum uE3 is low in Down syndrome pregnancy. 1987 Maternal serum hCG is elevated in Down syndrome pregnancy. 1988 Triple marker screening for Down syndrome in the second trimester is introduced. J. Canick, 2003

  10. Serum Screening Test Performanceat a fixed 5% False Positive Rate(Dating by Ultrasound) 100 80 69% 59% 60 DR at 5% FPR 37% 40 30% 20 0 AGE +AFP +hCG +uE3 single double triple 2nd trimester Wald et al. 2000 J. Canick, 2003

  11. New Approaches:Addition of Inhibin A J. Canick, 2003

  12. Maternal Serum Markers in the 2nd Trimester:Further improvement in screening for Down syndrome Background 1992 Maternal serum total inhibin is elevated in Down syndrome pregnancy 1994 Maternal serum inhibin A is elevated in Down syndrome pregnancy 1996 Inhibin A added to the triple test to make a quad marker test in the second trimester. Introduced in the UK at Barts. 1998Introduction of Quad Test in U.S. at Women & Infants’ J. Canick, 2003

  13. What is Inhibin A? • alpha-beta subunit glycoprotein hormone • inhibits the secretion of FSH from the anterior pituitary • synthesized in ovary and placenta (Inhibin B is synthesized in ovary and testis) • molecular weight approx. 32,000 daltons • inhibin-A = alpha subunit + betaA subunit Pro-a subunit Inhibin A bA subunit Inhibin B bBsubunit J. Canick, 2003

  14. Maternal Serum Inhibin A: Like hCG, it is, on average, about two times higher in Down syndrome than in unaffected pregnancies. Wald NJ et al, J Med Screen, 1997 J. Canick, 2003

  15. Similarity of hCG and inhA distributions in Down syndrome and unaffected pregnancy J. Canick, 2003

  16. 10 MoM 1 .1 Second Trimester Markers in 73 Down Syndrome Cases 1.91 2.00 0.74 0.61 AFP uE3 inhA hCG J. Canick, 2003

  17. Prenatal Screening Performance with Inhibin A J. Canick, 2003

  18. Serum Screening Test Performanceat a fixed 5% False Positive Rate(Dating by Ultrasound) 100 76% 80 69% 59% 60 DR at 5% FPR 37% 40 30% 20 0 AGE +AFP +hCG +uE3 +InhA single double triple quadruple 2nd trimester Wald et al. 2000 J. Canick, 2003

  19. Performance of the 2nd Trimester Quad Marker Test in Practice no. inhA DR FPR Study of DS med.MoM (%) (%) Barts program* 111 2.18 81 6.9 WIH program** 61 1.93 83 7.1 * 46,193 pregnancies screened over 5 years; 1 in 300 term risk cut-off used; Wald et al.,Lancet 2003 ** 40,450 pregnancies screened over 5 years; 1 in 380 term risk cut-off used; median MA = 28 years.; mss in preparation J. Canick, 2003

  20. Results of SURUSS (Serum, Urine, Ultrasound Study) PI: Nicholas Wald, FRCP, Wolfson Institute, London Objective: To determine the most effective, safe and cost- effective method of antenatal screening for Down’s syndrome using maternal age, nuchal translucency, and maternal serum and urine markers. Setting: 25 maternity units (24 in the UK) offering 2nd trimester serum screening that agreed to collect observational data in the 1st trimester. Size: results on 47,053 singleton pregnancies, including 101 with Down’s syndrome. J. Canick, 2003

  21. Results of SURUSS (Serum, Urine, Ultrasound Study) • Study Design: • Serum and Urine Markers: • 101 Downs, 505 controls • matched for gestational age and duration of sample storage • markers measured at 9-13 weeks gestation and at 14-22 weeks • Nuchal translucency: • All pregnancies • Intervention: • Based on on 2nd trimester screening • 1st trimester data observational J. Canick, 2003

  22. Results of SURUSS (Serum, Urine, Ultrasound Study) • Ability to compare 1st and 2nd trimester results: • Currently, the only study in the literature that can compare 1st and 2nd trimester screening fairly • The FASTER Trial, an NIH-sponsor study in the U.S. will also be able to compare 1st and 2nd trimester screening fairly • This is because there are two biases that must be taken into account in comparing the two time periods: • - Natural fetal loss in Down’s syndrome • - Marker related spontaneous fetal loss J. Canick, 2003

  23. Prediction SURUSS 100 81% 74% 80 66% 76% 69% 60 59% 42% DR at 5% FPR 40 37% 30% 20 0 AGE +AFP +hCG +uE3 +InhA single double triple quadruple 2nd trimester Serum Screening Test Performanceat a fixed 5% False Positive Rate Wald et al. 2000 J. Canick, 2003

  24. Mechanism? • Genes are not located on chromosome 21 • Tissue source? • Placenta • Fetal membranes • Fetus J. Canick, 2003

  25. Chromosome 21: Not the source of genes for serum markers identified so far. Marker chromosome AFP 4q hCG a 6q hCG b 19q inhibin a 2q inhibin bA 7p Chromosome 21 • 300 genes • 130 known human genes • Down syndrome critical region is 2-20 Mb in q22. • Likely that Down syndrome is a contiguous gene syndrome; unlikely that a single gene region is responsible. • Transcription factors, GF receptors, SOD1 on 21. J. Canick, 2003

  26. Down Syndrome Pregnancy Fetal Underproduction / Placental overproduction ? Fetal origin:Placental origin: AFP hCG DHEAS free a-subunit of hCG 16aOHDHEAS free b-subunit of hCG hPL Fetoplacental origin: SP1 estriol (unconj) inhibin-A (also total inhibin) estriol (conj) progesterone PLGF placental GH pro-MBP NAP (neutrophil alk phos) PAPP-A J. Canick, 2003

  27. Placental Inhibin A Subunit mRNA Levels Alpha : GAPDH Beta A : GAPDH Alpha Beta A Control Down syndrome Lambert-Messerlian et al., 1998 J. Canick, 2003

  28. New Approaches:The Integrated Test J. Canick, 2003

  29. Prenatal Screening for Down SyndromeA New Approach: The Integrated Test • Developed by Nicholas Wald in 1999. • The integration of the best tests performed at different times in pregnancy into a single test. • This will be more effective than current tests performed at any one time. 0 13 26 40 PAPP-A quad test = SERUM INTEGRATED NT+PAPP-A quad test= FULL INTEGRATED (weeks) Integrate results into a single risk J. Canick, 2003

  30. Detection rate at a fixed 5% false positive rate according to method of screening: Performance estimates based on modeling 100 90 80 70 60 Detection rate (%) 50 40 30 20 30% 37% 69% 76% 85% 85% 94% 10 0 MA + AFP triple quad combined serum full ------ 2nd trimester ------ 1st trim -- integrated -- PPV: 1:140 1:110 1:60 1:50 1:45 1:45 1:40 Wald et al, 1997, 1999, 2001 J. Canick, 2003

  31. Detection rate at a fixed 5% false positive rate according to method of screening: Modeling vs SURUSS ( ) 100 93% 89% 90 83% 81% 80 74% 70 60 Detection rate (%) 50 40 30 20 30% 37% 69% 76% 85% 85% 94% 10 0 MA + AFP triple quad combined serum full ------ 2nd trimester ------ 1st trim -- integrated -- PPV: 1:140 1:110 1:60 1:50 1:45 1:45 1:40 Wald et al, 1997, 1999, 2001, 2003 J. Canick, 2003

  32. False positive rate to achieve an 85% detection rate according to method of screening: Performance estimates based on modeling 20 14% 15 9.0% Fasle positive rate (%) 10 5.0% 5.0% 5 1.0% 0 triple quad combined serum full ---- 2nd trim ---- 1st trim -- integrated -- PPV: 1:130 1:85 1:45 1:45 1:9 Wald et al, 1997, 1999, 2001 J. Canick, 2003

  33. False positive rate to achieve an 85% detection rate according to method of screening: Performance estimates based on modeling 20 14% 15 9.0% Fasle positive rate (%) 10 5.0% 5.0% 5 1.0% 0 triple quad combined serum full ---- 2nd trim ---- 1st trim -- integrated -- PPV: 1:130 1:85 1:45 1:45 1:9 Wald et al, 1997, 1999, 2001 J. Canick, 2003

  34. False positive rate to achieve an 85% detection rate according to method of screening: Modeling vs SURUSS ( ) 20 15 10.9% Fasle positive rate (%) 10 7.1% 6.1% 5 3.0% 1.3% 14% 9.0% 5.0% 5.0% 1.0% 0 triple quad combined serum full ---- 2nd trim ---- 1st trim -- integrated -- PPV: 1:130 1:85 1:45 1:45 1:9 Wald et al, 1997, 1999, 2001, 2003 J. Canick, 2003

  35. False positive rate to achieve a 90% detection rate according to method of screening: modeling vs SURUSS ( ) 20 17.0% 15 11.7% 10.8% Fasle positive rate (%) 10 5.8% 5 2.8% 21.5% 15.2% 9.9% 8.8% 2.2% 0 triple quad combined serum full ---- 2nd trim ---- 1st trim -- integrated -- PPV: 1:190 1:135 1:90 1:90 1:20 Wald et al, 1997, 1999, 2001, 2003 J. Canick, 2003

  36. The Integrated Test • Advantages: • Safest and most effective screening policy • Substantially reduces the number of amnios needed • Preserves AFP screening for open NTD’s • Avoids confusion from several results • Stated Concerns: • Antenatal diagnosis and termination, if chosen, at about 16-17 instead of 13-14 weeks of pregnancy • 2-5 week waiting period before results are reported J. Canick, 2003

  37. New Approaches:Fetal Cells and Fetal DNA in theMaternal Circulation:Are They Useful in Prenatal Screeningfor Down Syndrome? J. Canick, 2003

  38. Fetal Cells Sorted from Maternal Blood Using Anti-Gamma Globin Gamma positive fetal cell pre-FISH Same cell post-FISH showing X and Y Courtesy of Dr. Diana Bianchi J. Canick, 2003

  39. Fetal Cells in the Maternal Circulation: History 1979fetal lymphocytesfirst fetal cells to be successfully enriched lymphocytesfrom maternal blood by the use of FACS (Herzenberg et al, PNAS 76:1453-5) 1990nucleated RBCs first study to enrich for fetal nucleated erythrocytes (Bianchi et al, PNAS USA 87: 3279-83) 1991nucleated RBCs first correct determination of fetal aneuploidy(Price et al, Am J Obstet Gynecol 165:1731-7) 1997nucleated RBCs increased number of fetal cells in blood of women with trisomy 21 fetuses (Bianchi et al, Am J Hum Genet 61:822-9) J. Canick, 2003

  40. Comparison of Down Syndrome Screening Performance of Fetal Cells and Second Trimester Maternal Serum test DR at 1.5% FPR fetal cells ~ 45% double screen ~ 40% triple screen ~ 50% quad screen ~ 60% Fetal cell data from Bianchi DW et al, Prenat Diagn 19: 993-7, 1999. Maternal serum data calculated from Wald NJ et al, J Med Screen 4:181-246, 1997. J. Canick, 2003

  41. Fetal DNA in the Maternal Circulation: History • cell-free DNA* circulating cell-free fetal DNA is also present in maternal blood (Lo et al, Lancet 350:485-7) • Method: Real time PCR analysis of maternal plasma or serum samples from pregnancies using • Y chromosome-specific probes. • 1999cell-free DNA* increased DNA concentrations in the plasma of pregnant women with trisomy 21 fetuses (Lo et al, Clin Chem 45:1747-50) • * Note that only DNA from a male fetus can be detected in maternal blood. J. Canick, 2003

  42. Fetal DNA in the Maternal Circulation: History • cell-free DNA* circulating cell-free fetal DNA is also present in maternal blood (Lo et al, Lancet 350:485-7) • Method: Real time PCR analysis of maternal plasma or serum samples from pregnancies using • Y chromosome-specific probes. • 1999cell-free DNA* increased DNA concentrations in the plasma of pregnant women with trisomy 21 fetuses (Lo et al, Clin Chem 45:1747-50) • * Note that only DNA from a male fetus can be detected in maternal blood. J. Canick, 2003

  43. Fetal DNA in the Maternal Circulation Are the levels higher in Down syndrome pregnancy? copies of fetal DNA/ml case/ plasma or serum (N) control study controls cases ratio comments Lo et al, 1999 Hong Kong 16 (18) 48 (6) 3.0 plasma / median Boston 23 (19) 46 (7) 2.0 plasma / median Zhong et al, 2000 83 (29) 186 (15) 2.2 plasma / mean Lee et al, 2002 24 (55) 41 (11) 1.7 serum / median Hromadnikova, 2002 25 (10) 23 (11) 0.9 plasma / median Spencer et al, 2003 34 (10) 32 (10) 0.9 serum / median Samura et al, 2001 32 (55) 24 (5) 0.8 serum / mean OVERALL (65) 1.64 J. Canick, 2003

  44. Fetal DNA in the Maternal Circulation Are the levels higher in Down syndrome pregnancy? copies of fetal DNA/ml case/ plasma or serum (N) control study controls cases ratio comments Lo et al, 1999 Hong Kong 16 (18) 48 (6) 3.0 plasma / median Boston 23 (19) 46 (7) 2.0 plasma / median Zhong et al, 2000 83 (29) 186 (15) 2.2 plasma / mean Lee et al, 2002 24 (55) 41 (11) 1.7 serum / median Hromadnikova, 2002 25 (10) 23 (11) 0.9 plasma / median Spencer et al, 2003 34 (10) 32 (10) 0.9 serum / median Samura et al, 2001 32 (55) 24 (5) 0.8 serum / mean OVERALL (65) 1.64 Small numbers! J. Canick, 2003

  45. Fetal DNA in the Maternal Circulation Are the levels higher in Down syndrome pregnancy? copies of fetal DNA/ml case/ plasma or serum (N) control study controls cases ratio comments Lo et al, 1999 Hong Kong 16 (18) 48 (6) 3.0 plasma / median Boston 23 (19) 46 (7) 2.0 plasma / median Zhong et al, 2000 83 (29) 186 (15) 2.2 plasma / mean Lee et al, 2002 24 (55) 41 (11) 1.7 serum / median Hromadnikova, 2002 25 (10) 23 (11) 0.9 plasma / median Spencer et al, 2003 34 (10) 32 (10) 0.9 serum / median Samura et al, 2001 32 (55) 24 (5) 0.8 serum / mean OVERALL (65) 1.64 Small effect? J. Canick, 2003

  46. 100 50 20 fetal DNA concentration (GE/ml) 10 15 16 17 18 19 Gestational Age (weeks) Levels of Fetal DNA in Maternal Serum in Women Carrying Male Euploid and Trisomy 21 Fetuses (Lee et al, Am J Obstet Gynecol 2002;187:1217-21) J. Canick, 2003

  47. Fetal Cells in the Maternal Circulation: Summary • Fetal cells in maternal blood is not yet a diagnostic test. • Fetal cell analysis is not yet as good as most serum screening methods for Down syndrome. • We must wait for advances in technology before fetal cells are useful in screening for diagnosis. • Fetal DNA in maternal blood appears to be elevated in Down syndrome pregnancy. • However, it is not yet useful in screening. • Further research is necessary, both to confirm the findings and to discover a method to quantify DNA which comes from female fetuses. J. Canick, 2003

  48. Second Trimester Inhibin A andNew Approaches in Prenatal Screening • Summary: • The goal in prenatal screening should be to provide the safest, most effective test to the greatest number of women. • Second trimester serum screening (double or triple markers) is effective, but is no longer the most effective screening method. • The addition of inhibin A improves 2nd trimester serum screening. The quad marker test should be offered to all women having screening beginning at 15 weeks gestation. J. Canick, 2003

  49. Second Trimester Inhibin A andNew Approaches in Prenatal Screening • Summary (continued): • First trimester combined screening (NT and serum markers) is an effective test for women who seek earlier diagnosis. • The Integrated Test is the safest, most effective test currently available. If NT measurement is not available, the Serum Integrated Test is the most effective method of serum screening. • Fetal cells or fetal DNA in the maternal circulation should be considered research, not to be applied clinically. J. Canick, 2003

  50. . .. llginize tesekkur ederim. Beni guzel ulkenize davet ettiginiz icin tesekkur ederim. .. .. .. J. Canick, 2003

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